Preservation of cell-free nucleic acids

ABSTRACT

A method for preserving and processing cell-free nucleic acids located within a blood sample is disclosed, wherein a blood sample containing cell-free nucleic acids is treated to reduce both blood cell lysis and nuclease activity within the blood sample. The treatment of the sample aids in increasing the amount of cell-free nucleic acids that can be identified and tested while maintaining the structure and integrity of the nucleic acids.

CLAIM OF PRIORITY

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 61/153,472, filed on Feb. 18, 2009, theentirety of the contents of that application being hereby incorporatedby reference for all purposes.

FIELD OF THE INVENTION

This invention relates to the identification and isolation of cell-freenucleic acids in blood samples and more particularly to the preservationof cell-free RNA within a blood sample.

BACKGROUND OF THE INVENTION

It has been recognized that mRNA obtained from plasma of a blood samplecan be useful as an indicator of protein expression. Thus, the presenceof extra-cellular or cell-free mRNA in blood plasma has triggered avariety of investigations aimed at determining the source and possiblediagnostic capabilities of these nucleic acids. Given that the blood ofmost healthy individuals ordinarily does not contain substantial amountsof cell-free RNA, elevated amounts of cell-free nucleic acids areusually indicative of a health issue (or pregnancy, as fetal cell-freenucleic acids have been identified in maternal blood). Specifically,elevated presence of cell-free mRNA has been found to indicate theexistence of various cancers thereby providing support for the beliefthat these nucleic acids may originate from tumor cells. Consequently,the identification of cell-free RNA within a blood sample could provideinsight into the presence and severity of cancer or some other condition(e.g., diabetes, inflammation, arthritis, infection, or the like), andmay thus be an early indicator of such condition. The identification ofcell-free RNA within a blood sample may also provide guidance for howbest to treat a patient depending upon the relative amounts of cell-freenucleic acids identified within the patient's blood sample. Thus it maybe useful not only for diagnosis, but also for patient treatment (e.g.,by evaluating a change in the RNA condition of a patient as treatmentprogresses).

RNA is typically subject to ribonuclease (RNase) activity that reducesthe amount of recoverable RNA from a blood sample. However, it has beendiscovered that despite the presence of ample RNase activity in bloodplasma, cell-free mRNA is unexpectedly very stable in vivo and avoidsany substantial nuclease mediated degradation. However, after a bloodsample is acquired from a patient, cell lysis begins and the nucleicacids from within the blood cells are mixed with the cell-free nucleicacids, making it difficult if not impossible to isolate and distinguishthe cell-free mRNA. Further, there is concern about the stability of thecell-free nucleic acids and their ability to avoid nuclease-initiateddegradation in vitro. Consequently, the disease indication capability ofthe cell-free nucleic acids may be diminished as their presence is nolonger accurately ascertainable. Ideally, prevention of cell lysis andcell-free nucleic acid degradation within the blood sample would allowfor the cell-free nucleic acids to be accurately measured and thepresence of any disease risk to be detected.

Efforts to further understand the unexpected stability of the cell-freenucleic acids in vivo have led to the belief that these nucleic acidsare able to avoid nuclease activity through protection from proteins orby being packaged into apoptotic bodies. In other words, as cellsundergo cell death or apoptosis, apoptotic bodies are produced and thecell-free nucleic acids become encased within a membrane of theapoptotic body, thereby reducing the susceptibility of the nucleic acidsto nucleases. However, there is concern that after blood draw thenucleic acids somehow become disassociated from the apoptotic bodies andbecome vulnerable to nucleases. There is a resulting need to process theblood samples containing the cell-free nucleic acids so that the nucleicacids continue to be unaffected by nucleases throughout processing toproduce accurate counts of cell-free nucleic acids for diagnosispurposes.

Metabolic inhibitors have been employed previously to inhibit metabolismin cells. For example, glyceraldehyde, sodium fluoride, and ATA, havebeen used to inhibit glucose metabolism in blood cells. See, e.g., U.S.Pat. Nos. 5,614,391; and 7,390,663 incorporated by reference herein. Theuse of formaldehyde-donor preservatives for cell or tissue, or RNApreservation has been described in U.S. Pat. Nos. 5,196,182; 5,260,048;5,459,073; 5,811,099; 5,849,517; and 6,337,189, incorporated byreference herein.

A number of patent documents address processes for the stabilizationand/or identification of nucleic acids located within blood plasma andtheir diagnostic applications. See, generally, U.S. Pat. Nos. 5,614,391;5,985,572; 6,617,170; 6,630,301; 6,759,217; 6,821,789; 6,916,634;6,939,671; 6,939,675; 7,208,275; 7,288,380, 7,569,350 and U.S. PatentPublication Nos. 2008/0057502; 2008/0096217; and 2008/0261292 allincorporated by reference herein. Notwithstanding the above, thereremains a need for cell-free RNA isolation and preservation methods thatpreserve cell-free RNA substantially as it exists at the time of a blooddraw in an effort to maximize the amount of recovered cell-free nucleicacid from blood plasma and produce reliable isolation and diagnosticresults.

The present invention addresses the need for an efficient and consistentmethod of preserving and testing of a blood sample for elevated levelsof cell-free RNA in plasma of the blood sample, which unexpectedly andsurprisingly results in short term inhibition of metabolism (i.e., RNAsynthesis); long term fixing of blood cells of the blood sample toprevent leaking of cellular RNA into the plasma; fixing the cellular RNAthat is within the blood cells to freeze (e.g., immobilize) the proteinexpression pattern of the blood cells; and stabilizing and protectingthe RNA that is in the plasma from nucleases and proteases.

SUMMARY OF THE INVENTION

The present invention contemplates a screening method for theidentification of a disease state, comprising the steps of: contacting adrawn blood sample that includes a plurality of blood cells with aplasma RNA (e.g., mRNA) protective agent; isolating cell-free RNA fromthe blood sample; and analyzing (e.g., by quantity, quality, or both)the isolated RNA for the presence, absence, or severity of a diseasestate. The protective agent may be present in an amount and for a timesufficient so that the RNA synthesis is inhibited for at least twohours. The protective agent may be present in an amount so that bloodcells of the drawn blood sample are fixed to substantially preventleaking of cellular RNA into the plasma. The protective agent may bepresent in an amount so that any cellular RNA that is within the bloodcells at the time of the blood draw is substantially preserved toimmobilize the protein expression pattern of the blood cells so that theprotein expression pattern of the cells remains substantially the sameas at the time of the blood draw. The protective agent may be present inan amount so that the RNA that is in the plasma is substantiallystabilized against degradation mediated by the combined action ofnucleases and proteases.

The protective agent may include one or more preservative agents, one ormore enzyme inhibitors, one or more metabolic inhibitors, or anycombination thereof. The one or more preservative agents may include aformaldehyde releaser such as one selected from the group consisting of:diazolidinyl urea, imidazolidinyl urea,dimethoylol-5,5dimethylhydantoin, dimethylol urea,2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo [3.3.0]octane,5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane,quaternary adamantine and any combination thereof. The one or moreenzyme inhibitors may be selected from the group consisting of: diethylpyrocarbonate, ethanol, aurintricarboxylic acid (ATA), glyceraldehydes,sodium fluoride, ethylenediamine tetraacetic acid (EDTA), formamide,vanadyl-ribonucleoside complexes, macaloid, heparin,hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate,dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol,tris (2-carboxyethyl) phosphene hydrochloride, a divalent cation such asMg⁺², Mn⁺², Zn⁺², Fe⁺², Ca⁺², Cu⁺² and any combination thereof. The oneor more metabolic inhibitors may be selected from the group consistingof: glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate,2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glyceratedihydroxyacetate, sodium fluoride, K₂C₂O₄ and any combination thereof.

The concentration of the preservative agent prior to the contacting stepmay be at least about 50 g/l. The concentration of the preservativeagent prior to the contacting step may be less than about 500 g/l. Theconcentration of the preservative agent prior to the contacting step maybe at least about 200 g/l. The concentration of the preservative agentprior to the contacting step may be less than about 300 g/l. Theconcentration of the preservative agent prior to the contacting step maybe a concentration at which cross-linking of nucleic acids and proteinsis observed, as indicated by agarose gel electrophoresis.

The isolating step may include isolating the nucleic acid from plasma ofthe blood sample. Either or both of the isolating and analyzing stepsmay occur at least 2 hours, 7 days, or even 14 days after the bloodsample is drawn. Either or both of the isolating and analyzing steps mayoccur without freezing the blood sample (e.g. to a temperature colderthan about −30° C. (more preferably colder than about −70° C.)). Theanalyzing step, the isolating step or both may include a step ofcontacting the nucleic acid with an enzyme, an amplifier or both.

The contacting step may take place in a blood collection tube into whichthe blood sample is drawn. The contacting step may take place as theblood sample is drawn. The contacting step may be sufficient so thatafter a period of at least 7 days from the time the blood sample isdrawn, the amount of cell-free RNA present in the blood sample is atleast about 90%, at least about 95%, or about 100% of the amount ofcell-free RNA present in the blood sample at the time the blood sampleis drawn. The contacting step may be sufficient so that after a periodof at least about 7 days from the time the blood sample is drawn, theconcentration of cell-free RNA relative to the total nucleic acid in theblood sample that is present is at least about 10 times, at least about20 times, or at least about 50 times the amount of cell-free RNA thatwould be present in the absence of the contacting step.

The protective agent may include a metabolic inhibitor selected from thegroup consisting of: glyceraldehyde, dihydroxyacetone phosphate,glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate,2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glyceratedihydroxyacetate, sodium fluoride, K₂C₂O₄ and any combination thereof.The protective agent may include a protease inhibitor selected from thegroup consisting of: antipain, aprotinin, chymostatin, elastatinal,phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin,soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, pepstatin,VdLPFFVdL, EDTA, 1,10-phenanthroline, phosphoramodon, amastatin,bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima beantrypsin inhibitor, pancreatic protease inhibitor, egg white ovostatinegg white cystatin, and any combination thereof. The protective agentmay include a phosphatase inhibitor selected from the group consistingof: calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaicacid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin,cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilincomplexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen),dephostatin, mpV(pic) DMHV, sodium orthovanadate and any combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation showing the glucose concentrationpresent within blood samples contacted with six different compositionsover time.

FIG. 2 is a graphic representation of showing the relative amounts ofcell-free RNA present within four blood samples over time using the 18SrRNA copy number as a marker where an increase in the 18S rRNA copynumber is indicative of cell lysis or increased cell metabolism; thereis seen a plot of “preservative agent+nuclease inhibitor+metabolicinhibitor 1 & 2” that shows constant concentration of 18S rRNA that isindicative of uncontaminated (from cellular mRNA) and protected (fromnuclease mediated degradation) cell-free mRNA.

FIG. 3 is a graphic representation showing the relative amounts ofcell-free RNA present within two blood samples over time using theRASSF1A copy number as a marker.

DETAILED DESCRIPTION

In general, the invention herein contemplates a screening method for theidentification of disease presence which includes the preservation andisolation of cell-free nucleic acids located within a blood sample. Aunique preservation step acts to increase the amount of recoverablenucleic acids thereby improving the diagnostic capabilities of thecell-free nucleic acids.

The present invention provides a method for the isolation of nucleicacids using a protective agent. The nucleic acid may be DNA, RNA, or anycombination thereof. The nucleic acid may be cell-free DNA, cell-freeRNA, or any combination thereof. The samples from which the nucleicacids may be isolated include any blood sample. The cell-free nucleicacids may be located within plasma. The method disclosed herein allowsfor the efficient preservation and isolation of cell-free (e.g.,extra-cellular) nucleic acids while avoiding possible mixing withnucleic acids originating within the blood cells that enter a bloodsample due to cell lysis after blood draw.

The process for improved cell-free nucleic acid isolation from a bloodsample begins by contacting a blood sample with a protective agentcontaining one or more active ingredients to maintain the integrity ofthe components within the sample. The one or more active ingredients mayinclude a preservative agent. The preservative agent may include aformaldehyde donor composition. Ingredients that may be used as apreservative agent include, but are not limited to, diazolidinyl urea,imidazolidinyl urea, dimethoylol-5,5dimethylhydantoin, dimethylol urea,2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane,5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo [3.3.0]octane,quaternary adamantine or any combination thereof. The preservative agentmay be selected from the group consisting of diazolidinyl urea (DU),imidazolidinyl urea (IDU), and any combination thereof.

The amount of preservative agent used is generally about 50 to about 500grams per liter. For example, the preservative agent may comprise about200 to about 300 grams of DU per liter of buffered salt solution.

As used throughout the present teachings, the protective agentcomposition preferably is substantially non-toxic. For example, themethods herein (and compositions used herein) are free of separatelyadding and/or handling of any materially significant concentration(e.g., less than about 1% by weight, more preferably less than about2000 parts per million, more preferably less than about 1000 parts permillion, and still more preferably less than about 500 parts permillion) of formaldehyde and/or paraformaldehyde prior to any contactwith a blood product sample. Further, the protective agent may besubstantially free of guanidinium salts, sodium dodecyl sulfate (SDS),or any combination thereof.

The protective agent may further contain one or more nuclease inhibitors(e.g., enzyme inhibitors) in a suitable amount to prevent DNase and/orRNase activity from decreasing the quality and amount (e.g. by at leastabout 10% by weight, and more preferably at least about 50% by weight)of cell-free nucleic acids recoverable from the blood sample as comparedwith a sample that does not include a nuclease inhibitor. Nucleaseinhibitors that may be used include, but are not limited to diethylpyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide,vanadyl-ribonucleoside complexes, macaloid, ethylenediamine tetraaceticacid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion,bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol,cysteine, dithioerythritol, tris (2-carboxyethyl) phosphenehydrochloride, or a divalent cation such as Mg⁺², Mn⁺², Zn⁺², Fe⁺²,Ca⁺², Cu⁺² and any combination thereof. More preferably, the nucleaseinhibitors that may be used include aurintricarboxylic acid (ATA),ethylenediamine tetraacetic acid (EDTA), and any combination thereof.Preferred nuclease inhibitors may bind to nucleases (e.g., RNases) sothat the nucleases are no longer capable of contacting the cell-free RNAthereby reducing the adverse effects of nucleases on the quantity andquality of the cell-free RNA. The one or more nuclease inhibitors may bepresent in an amount sufficient to prevent nuclease activity fromreducing the amount of recoverable cell-free RNA by more than about 15%.

The protective agent may also include one or more metabolic inhibitorsin a suitable amount to reduce cell metabolism within a blood sample.Metabolic inhibitors that may be used include, but are not limited toglyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate,1,3-bisphosphoglycerate, 3-phosphoglycerate, 2- phosphoglycerate,phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodiumfluoride, K₂C₂O₄ and any combination thereof. More preferably, the oneor more metabolic inhibitors used may include sodium fluoride,glyceraldehyde and any combination thereof. Preferred metabolicinhibitors may reduce degradation of cell-free RNA and also reduce celllysis so that cellular RNA does not become intermixed with any cell-freeRNA. This intermixing of cell-free RNA and cellular RNA may reduce theaccuracy of any measurement of the amount of cell-free RNA in a bloodsample. As an example, in the event that severity of a specified cancermay be measured by the amount of cell-free RNA in a blood sample, anysample that has not been treated to inhibit metabolism may show anunusually high amount of cell-free RNA, even though much of thatcell-free RNA originated within one or more blood cells. Thus, the testresult may show a false positive of increased cancer severity when infact the actual amount of true cell-free RNA was low, representing acancer of reduced severity. The one or more metabolic inhibitors may bepresent in an amount sufficient to prevent cell metabolism from reducingthe accuracy of any cell-free RNA measurement by more than about 15%.

The protective agent may also include one or more protease inhibitingcompounds which may limit RNA synthesis. Such protease inhibitingcompounds may include but are not limited to antipain, aprotinin,chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF,TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid(IAA), E-64, pepstatin, VdLPFFVdL, EDTA, 1,10-phenanthroline,phosphoramodon, amastatin, bestatin, diprotin A, diprotin B,alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic proteaseinhibitor, egg white ovostatin, egg white cystatin and any combinationthereof. Combinations of protease inhibitors, commonly referred to as a“protease inhibition cocktail” by commercial suppliers of suchinhibitors, may also be used within the protective agent. Such“cocktails” are generally advantageous in that they provide stabilityfor a range of proteins of interest. Preferred protease inhibitors mayinclude aprotonin, EDTA, EGTA, PMSF, and any combination thereof. Theone or more protease inhibiting compounds may be present in an amountsufficient to prevent RNA synthesis from reducing the accuracy of anycell-free RNA measurement by more than about 15%.

The protective agent may further include one or more phosphataseinhibitors including but not limited to calyculin A, nodularin, NIPP-1,microcystin LR, tautomycin, okadaic acid, cantharidin, imidazole,microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin,endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes,cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin,mpV(pic) DMHV, sodium orthovanadate and combinations thereof.Phosphatase inhibitor cocktails may also be included within theprotective agent, as they also provide stability for a wide range ofproteins. Preferred phosphatase inhibitors may include cantharidin,sodium orthovanadate, imidazole and any combination thereof. The one ormore phosphatase inhibiting compounds may be present in an amountsufficient to prevent a reduction in the accuracy of any cell-free RNAmeasurement by more than about 15%.

The protective agent may include one or more polyamines in a suitableamount such that they are capable of binding with any nucleic acidsthereby preventing degradation of the nucleic acids. The polyamines thatmay be added include but are not limited to spermine, spermidine,putrescine, cadaverine, and combinations thereof. Preferably, thepolyamines used may include spermine and spermidine.

The initial contacting of the blood sample with the protective agent maybe for a time sufficient to inhibit cell lysis, nuclease activity, orany combination thereof. Contacting may occur for at least about 10seconds, at least about 1 minute, or at least about 2 minutes.Contacting can occur for longer periods of time. For example, contactingmay be commenced substantially contemporaneously from the time of blooddraw (e.g., within less than about 10 minutes of the blood draw) and itmay last until nucleic acids are isolated, screened, and/or tested. Thecontacting step may also be employed to provide a sample with a longershelf life. Thus, it is possible that a lapse of time of at least about2 hours, more preferably at least about 6 hours, at least about 24hours, at least about 7 days or even at least about 14 days can elapsebetween the time of blood draw (which may be substantiallycontemporaneous with the contacting step), and the time of any testingor screening of the sample, and or isolation of the nucleic acids.

The protective agent may comprise an active agent in solution. Suitablesolvents include water, saline, dimethylsulfoxide, alcohol and anymixture thereof. The protective agent solution may comprise diazolidinylurea (DU) and/or imidazolidinyl urea (IDU) in a buffered salt solution.The protective agent solution may further comprise EDTA and ATA. Theprotective agent solution may also include one or more metabolicinhibitors. The protective agent solution may contain only a fixativeand may be substantially free of any additional additives.

The amount of any active ingredient within the protective agent maygenerally be at least about 0.01% by weight. The amount of any activeingredient within the protective agent may generally be less than about70% by weight. The protective agent may comprise at least about 10%diazolidinyl urea. The protective agent may comprise less than about 40%diazolidinyl urea. The protective agent may further contain at leastabout 1% of one or more enzyme inhibitors (e.g., nuclease inhibitors)such as EDTA and ATA. The protective agent may contain less than about30% of one or more enzyme inhibitors. The protective agent may alsocontain at least about 1% of one or more metabolic inhibitors. Theprotective agent may contain less than about 20% of one or moremetabolic inhibitors.

The amount of preservative agent (e.g., fixative) relative to the amountof any enzyme inhibitors (e.g., EDTA and ATA) is preferably about 1 toabout 10 parts (more preferably about 3 to about 6 parts) by weight offixative to about 2 parts by weight of enzyme inhibitors. The amount offixative (e.g. the formaldehyde releaser) relative to the amount of anyone or more metabolic inhibitors is preferably about 1 to about 10 parts(more preferably about 3 to about 7 parts) by weight of fixative toabout 1 part by weight metabolic inhibitors. The amount of protectiveagent within a tube prior to blood draw may be at least about 200g/liter. The amount of protective agent within a tube prior to blooddraw may be less than about 1000 g/liter.

The combination of one or more preservative agents (e.g. theformaldehyde releasers), one or more enzyme inhibitors, one or morenuclease inhibitors and one or more metabolic inhibitors within theprotective agent results in improved ability to maintain the amount andquality of cell-free RNA within a blood sample and is used in a mannerand amount so that such results are obtained. These results are believedunexpected and superior to results obtained by the use of only thepreservative agent, only the enzyme inhibitor, only the nucleaseinhibitor, only the metabolic inhibitor or any combination including twoof the preservative agent, the enzyme inhibitor, the nuclease inhibitor,or the metabolic inhibitor. Therefore, it can be appreciated that asynergistic effect occurs when a preservative agent, enzyme inhibitor,nuclease inhibitor, and metabolic inhibitor are combined.

The protective agent can be located within a specialized device, whereinthe protective agent is already present in the device prior to additionof the blood sample, such as that disclosed in U.S. Patent PublicationNo. 2004/0137417, incorporated by reference herein. More preferably, thedevice is an evacuated collection container, usually a tube. The tubemay preferably be made of a transparent material that will also resistadherence of the cells within a given sample. The interior wall of thetube may be coated or otherwise treated to modify its surfacecharacteristics, such as to render it more hydrophobic and/or morehydrophilic, over all or a portion of its surface. The tube may have aninterior wall flame sprayed, subjected to corona discharge, plasmatreated, coated or otherwise treated. The tube may be treated bycontacting an interior wall with a substance so that the nucleic acidsof interest will resist adhering to the tube walls. The surface of thetube may be modified to provide a dual functionality that simultaneouslyprovides an appropriate balance of desired hydrophilicity andhydrophobicity, to allow collection of blood, dispersion of thepreservatives herein, and resistance of adhesion of nucleic acids to theinner wall of a blood collection tube. Thus it is possible that anycoating may be a functionalized polymeric coating that includes a firstpolymer and one or more second monomeric and/or polymericfunctionalities that are different from (e.g., chemically differentfrom) the first polymer. The coating may include one or more co-polymers(e.g., block copolymer, graft copolymer, or otherwise). For example, itmay include a copolymer that includes a first hydrophobic polymericportion, and a second hydrophilic polymeric portion. The coating may bea water based coating. The coating may optionally include an adhesionpromoter. The coating may be applied in any suitable manner, it may besprayed, dipped, swabbed, or otherwise applied onto some or all of theinterior of the blood collection tube. The coating may also be appliedin the presence of heat. Preferably any coating applied to the innerwall of a blood collection tube will form a sufficiently tenacious bondwith the glass (e.g., borosilicate glass) or other material (e.g.,polymeric material) of the tube so that it will not erode or otherwiseget removed from the inner wall. Examples of suitable polymeric coatingsmay include silicon containing polymers (e.g., silanes, siloxanes, orotherwise); polyolefins such as polyethylene or polypropylene;polyethylene terephthalate; fluorinated polymers (e.g.,polytetrafluoroethylene); polyvinyl chloride, polystyrene or anycombination thereof. Examples of teachings that may be employed to coatan interior of a blood collection tube may be found in U.S. Pat. Nos.6,551,267; 6,077,235; 5,257,633; and 5,213,765; all incorporated byreference.

The composition included in the tube may be present in an amountsufficient to preserve cell morphology and prevent cell degradationwhile also preventing deleterious DNase and RNase activity within thecell-free nucleic acids. However, the amount may also be sufficientlysmall so that any consequential dilution of the sample is substantiallyavoided, and cell-free nucleic acids in the sample are not materiallydiluted. A blood sample may be fixed simultaneously as it is drawn intothe specialized tube. The tube may also be coated over an exterior wallwith a protective coating (e.g., a containment barrier that helpscontrol glass shard fragmentation) such as that disclosed in U.S. Pat.No. 7,419,832, incorporated by reference herein.

Additionally, the protective agent may be in a highly viscous orsubstantially solid state, such that (for example) it can be usedeffectively as a substantially solid state coating. Examples of suchsubstantially solid state preservatives can be found in commonly ownedco-pending U.S. patent application Ser. No. 12/646,204, incorporated byreference herein. Liquid removal techniques can be performed on theprotective agent in order to obtain a substantially solid stateprotective agent. Liquid removal conditions may preferably be such thatthey result in removal of at least about 50% by weight, more preferablyat least about 75% by weight, and still more preferably at least about85% by weight of the original amount of the dispensed liquid protectiveagent. Liquid removal conditions may preferably be such that they resultin removal of sufficient liquid so that the resulting composition is inthe form of a film, gel or other substantially solid or highly viscouslayer; for example it may result in a substantially immobile coating(preferably a coating that can be re-dissolved or otherwise dispersedupon contact with a blood product sample). Thus, liquid removalconditions may preferably be such that they result in a material thatupon contact with the sample under consideration (e.g., a maternal bloodsample) the protective agent will disperse in the sample, andsubstantially preserve components (e.g., cell-free nucleic acids) in thesample. Liquid removal conditions may preferably be such that theyresult in a remaining composition that is substantially free ofcrystallinity; has a viscosity that is sufficiently high that theremaining composition is substantially immobile at ambient temperature(e.g., it does not exhibit any visibly detectable (as seen by the nakedeye) flow when held in a storage device at room temperature on anincline of at least about 45° for at least one hour); or both. In thisregard as taught in the forgoing application a colorant may also beemployed.

As discussed herein, contacting a blood or plasma sample with theprotective agent allows the sample to be stored for a period of timeprior to isolating and testing the nucleic acids. A blood or plasmasample may be drawn at one location (e.g., a health care facility),contacted with the protective agent, and later transported to adifferent remote location (e.g., a laboratory, such as one that isseparately housed at a distance of at least about 1 km, 2 km, 3 km, orfurther away from the draw site) for the nucleic acid isolation andtesting process. The nucleic acids can be isolated from the blood orplasma sample and tested at the remote location and the resultingdiagnostic information may be reported to the site of the original blooddraw. The nucleic acid isolation process may be performed at one remotelocation and the resulting data can be analyzed to identify thepresence, absence or relative severity of a disease state at a thirdlocation. Alternatively, the results of the cell-free nucleic acidisolation process may be sent back to the site of the initial blood drawand analyzed there. The resulting diagnostic information may then besent to a third location or back to the remote location or the site ofthe initial blood draw.

At any time after the initial contact of the blood or plasma sample withthe protective agent, the sample can be treated to isolate the cell-freenucleic acids located within the blood. The nucleic acids may beisolated using any isolation method including those methods disclosed incommonly owned U.S. Patent Publication No. 2009/0081678, incorporated byreference herein. The protective agent may aid in maintaining theintegrity of blood cell membranes (e.g., the cell membranes remainintact), so that nucleic acids are not released into the sample fromblood cells having ruptured cell membranes. Any cell membrane rupturemay cause cellular nucleic acids to enter the plasma making isolation ofthe cell-free nucleic acids (e.g., identifying and separating cell-freenucleic acids from nucleic acids that originated within a blood cell)more difficult. The fixative agent may act to prevent cell lysis so thatthe blood cells remain intact and substantially all cellular nucleicacids remain intra-cellular to avoid unwanted contamination of thecell-free nucleic acids.

After the cell-free nucleic acids have been isolated, they can be testedto identify the presence, absence or severity of a disease stateincluding but not limited to a multitude of cancers. The methods hereinthus further contemplate a step of nucleic acid testing. Testing of thenucleic acids can be performed using any nucleic acid testing methodincluding, but not limited to polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), quantitative real timepolymerase chain reaction (Q-PCR), gel electrophoresis, capillaryelectrophoresis, mass spectrometry, fluorescence detection, ultravioletspectrometry, DNA hybridization, allele specific polymerase chainreaction, polymerase cycling assembly (PCA), asymmetric polymerase chainreaction, linear after the exponential polymerase chain reaction(LATE-PCR), helicase-dependent amplification (HDA), hot-start polymerasechain reaction, intersequence-specific polymerase chain reaction (ISSR),inverse polymerase chain reaction, ligation mediated polymerase chainreaction, methylation specific polymerase chain reaction (MSP),multiplex polymerase chain reaction, nested polymerase chain reaction,solid phase polymerase chain reaction, or any combination thereof.

The present invention includes a method for isolating and testingcell-free RNA from plasma. The method can be performed on a singlesample or on multiple samples simultaneously (e.g., in a multi-wellplate). The method includes contacting a plasma sample with a protectiveagent. The protective agent may include one or more active ingredientsas previously discussed so that the blood cells remain intact throughoutthe blood draw and RNA isolation process. The protective agent mayfurther include one or more RNase or enzyme inhibitors and one or moremetabolic inhibitors to maintain the structural integrity of the RNA.After contacting the blood sample with the protective agent, the samplemay be centrifuged to separate the plasma. The cell pellet may then bediscarded. Alternatively, by contacting a blood sample with theprotective agent, the blood sample does not necessarily requireimmediate processing and can be stored for up to about 14 days at roomtemperature. Thus the inventions herein contemplate one or more steps ofstoring and/or otherwise waiting a relatively lengthy period from thetime of blood draw and/or contacting until the time of screening,testing or other analysis. Once the sample is processed, an appropriateconcentration of salt and alcohol may be added to precipitate the RNAcontaining material. An organic or other compound such as a phenolderivative or the like may then be added to remove any remaining proteincontaminants. Any protein contaminants that still remain may be removedby adding additional amounts of an organic or other compound such as aphenol derivative or the like. After centrifugation, ethanol may beadded and the sample centrifuged again. Any remaining liquid may beremoved from the sample so only the RNA will remain. The finishedproduct of isolated RNA may then be contacted with a buffer.

Incubation may also occur. For example, incubation may occur on ice orat any temperature between −30° C. and 70° C. A sample may be incubatedat about −20° C. A sample may also be stored at room temperature andthus substantially free of any freezing upon blood draw.

Preferably, centrifuging occurs at speeds of about 500 to about 15,000rpm. Centrifuging may occur at about 1,000 to 13,000 rpm. Centrifugingmay be performed at a temperature of about 1-20° C. Centrifuging may beperformed at a temperature of about 4-9° C.

Example 1

Blood samples from the same donor are drawn into six separate bloodcollection tubes (tube 1 through tube 6). Tube 1 contains only EDTA.Tube 2 contains DU and EDTA. Tube 3 contains DU, EDTA and ATA. Tube 4contains DU, EDTA, ATA and glyceraldehyde. Tube 5 contains DU, EDTA, ATAand sodium fluoride. Tube 6 contains DU, EDTA, ATA, glyceraldehyde andsodium fluoride. All tubes are stored at room temperature and 1 mlaliquots of blood are removed from each tube at hours 1.5,8, 24, 48, 72and 96. The blood glucose levels of each sample are measured using a YSIblood glucose meter available from YSI Life Sciences (Yellow Springs,Ohio). The blood glucose concentration of those samples from tube 6maintained relatively consistent glucose levels over the test period,indicating that the combination of EDTA, DU, ATA, glyceraldehyde andsodium fluoride provided reduced levels of cell metabolism. The resultsof this example are shown in a graphic format at FIG. 1.

Example 2

Four blood samples from the same donor are drawn into four separateblood collection tubes, tube A through tube D. Tube A contains DU, EDTA,ATA, glyceraldehyde and sodium fluoride. Tube B contains DU, EDTA andATA. Tube C contains DU and EDTA. Tube D contains only EDTA. All tubesare stored at room temperature and 1 ml aliquots of blood are removedfrom each tube on day 0, day 1, day 2, and day 3 and plasma isseparated. All samples are centrifuged at 800 g for 10 minutes at roomtemperature to separate the plasma. The plasma is then transferred intonew tubes and centrifuged at 1500 g for 10 minutes at room temperature.Free circulating RNA is purified using the QIAamp MinElute Virus Spinkit available from Qiagen, Inc. (Valencia, Calif.). RNA is extractedfrom each plasma sample. The samples are then amplified by Real Time PCR(using TaqMan® RT PCR reagents available from Applied Biosystems, FosterCity, Calif.) to identify the 18S rRNA copy number per ml of plasma.Results showed a consistent relative percentage of 18S rRNA copy numberper ml of plasma (about 0%) at each measurement, indicating little or nocellular RNA presence as a result of cell lysis or increased cellmetabolism in the plasma samples originating in Tube A (containing DU,EDTA, ATA, glyceraldehyde and sodium fluoride). The 18S rRNA copy numberper ml of plasma showed elevated levels at every measurement in thosesamples originating in tubes B, C, and D indicating an increase in theamount of cellular RNA present as a result of cell lysis or increasedcell metabolism. The results of this example are shown in a graphicformat at FIG. 2.

Example 3

Blood samples from the same donor are drawn into two separate bloodcollection tubes. The first tube contains DU, EDTA, ATA, glyceraldehydeand sodium fluoride. The second tube contains only EDTA. Both tubes arestored at room temperature and 1 ml aliquots of blood are removed fromeach tube on day 0, day 1, day 2, day 7, and day 8 and plasma isseparated. All samples are centrifuged at 800 g for 10 minutes at roomtemperature to separate the plasma. The plasma is then transferred intonew tubes and centrifuged at 1500 g for 10 minutes at room temperature.Free circulating RNA is purified using the QIAamp MinElute Virus Spinkit available from Qiagen Inc. (Valencia, Calif.). RNA is extracted fromeach plasma sample. The samples are then amplified by Real Time PCR(using TaqMan® RT PCR reagents available from Applied Biosystems, FosterCity, Calif.) to identify fragments of β-globin and RASSF1A genes withinthe plasma. Results showed a consistent relative percentage of RASSF1Agenes per ml of plasma at each measurement, indicating little decreaseof RNA presence in the plasma samples originating in the first tube(containing DU, EDTA, ATA, glyceraldehyde and sodium fluoride). TheRASSF1A genes per ml of plasma showed decreased levels at everymeasurement in those samples originating in the tube containing onlyEDTA indicating a decrease in the amount of cell-free RNA present overtime. The results of this example are shown in a graphic format at FIG.3.

Examples 1, 2 and 3 above demonstrate an unexpected synergistic effectoccurring only in blood samples contacted by a fixative, one or moreenzyme inhibitors (e.g., nuclease inhibitors), and one or more metabolicinhibitors or more specifically, by a protective agent including DU,EDTA, ATA, glyceraldehyde and sodium fluoride. Blood samples contactedby only a fixative, only an enzyme inhibitor, only a metabolic inhibitoror any combination including less than all of the above components donot demonstrate the ability to maintain the integrity of the blood cellsor the integrity of the nucleic acids. The combined effect of the DU,EDTA, ATA, glyceraldehydes and sodium fluoride far exceeds anyexpectations based on the effect, or lack thereof, of the DU, EDTA, ATA,glyceraldehydes or sodium fluoride used alone.

It will be appreciated that concentrates or dilutions of the amountsrecited herein may be employed. In general, the relative proportions ofthe ingredients recited will remain the same. Thus, by way of example,if the teachings call for 30 parts by weight of a Component A, and 10parts by weight of a Component B, the skilled artisan will recognizethat such teachings also constitute a teaching of the use of Component Aand Component B in a relative ratio of 3:1. Teachings of concentrationsin the examples may be varied within about 25% (or higher) of the statedvalues and similar results are expected. Moreover, such compositions ofthe examples may be employed successfully in the present methods toisolate nucleic acids (e.g., cell-free RNA).

It will be appreciated that the above is by way of illustration only.Other ingredients may be employed in any of the compositions disclosedherein, as desired, to achieve the desired resulting characteristics.Examples of other ingredients that may be employed include antibiotics,anesthetics, antihistamines, preservatives, surfactants, antioxidants,unconjugated bile acids, mold inhibitors, nucleic acids, pH adjusters,osmolarity adjusters, or any combination thereof.

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

1-16. (canceled) 17: A direct blood draw tube for stabilization ofcell-free nucleic acid within a blood sample comprising: a compositionincluding: a) one or more formaldehyde releaser preservative agents; b)one or more metabolic inhibitors; c) one or more protease inhibitors; d)ethylenediaminetetraacetic acid (EDTA); and e) one or more solvents;wherein the composition is free of separately added formaldehyde;wherein the amount of composition within the direct blood draw tubeprior to blood draw is less than about 1000 g/liter; and wherein thedirect blood draw tube including the composition and a blood samplelocated therein is storable at room temperature for at least about 14days without cell lysis and cell-free nucleic acid degradation of theblood sample. 18-20. (cancelled) 21: The direct draw blood tube claim17, wherein the one or more formaldehyde releaser preservative agentsinclude imidazolidinyl urea. 22: The direct draw blood tube of claim 17,wherein the one or more formaldehyde releaser preservative agents areselected from the group consisting of: diazolidinyl urea, imidazolidinylurea, dimethoylol-5,5dimethylhydantoin, dimethylol urea,2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo [3.3.0]octane,5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane,quaternary adamantine and any combination thereof. 23: The direct drawblood tube of claim 17, wherein the one or more metabolic inhibitors areselected from the group consisting of: glyceraldehyde, dihydroxyacetonephosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate,3-phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, pyruvate,glycerate dihydroxyacetate, sodium fluoride, K₂C₂O₄ and any combinationthereof. 24: The direct draw blood tube of claim 17, wherein the one ormore protease inhibitors are selected from the group consisting of:antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonylfluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsininhibitor, indoleacetic acid (IAA), E-64, pepstatin, VdLPFFVdL, EDTA,1,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A,diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor,pancreatic protease inhibitor, egg white ovostatin egg white cystatin,and any combination thereof. 25: The direct draw blood tube of claim 22,wherein the one or more metabolic inhibitors are selected from the groupconsisting of: glyceraldehyde, dihydroxyacetone phosphate,glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate,2-phosphoglycerate, phosphoenolpyruvate, pyruvate, glyceratedihydroxyacetate, sodium fluoride, K₂C₂O₄ and any combination thereof.26: The direct draw blood tube of claim 25, wherein the one or moreprotease inhibitors are selected from the group consisting of: antipain,aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride(PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor,indoleacetic acid (IAA), E-64, pepstatin, VdLPFFVdL, EDTA,1,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A,diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor,pancreatic protease inhibitor, egg white ovostatin egg white cystatin,and any combination thereof. 27: The direct draw blood tube of claim 17,wherein the direct blood draw tube is comprised of a polymeric materialand/or an interior wall of the tube is coated or otherwise treated witha polymeric coating to modify its surface characteristics. 28: Thedirect draw blood tube of claim 27, wherein the polymeric materialincludes polyethylene terephthalate. 29: The direct draw blood tube ofclaim 24, wherein the direct draw blood tube includes imidazolidinylurea as a formaldehyde releaser preservative agent and sodium fluorideas a metabolic inhibitor. 30: The direct blood draw tube of claim 28,wherein the composition is in a form of a gel or other substantiallysolid or highly viscous layer and wherein the composition isre-dissolved or otherwise dispersed upon contact with the blood sample.31: A direct blood draw tube for stabilization of cell-free nucleic acidwithin a blood sample comprising: a composition including: a)imidazolidinyl urea; b) one or more enzyme inhibitors; wherein the oneor more enzyme inhibitors are selected from the group consisting of:diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA),glyceraldehydes, sodium fluoride, ethylenediaminetetraacetic acid(EDTA), formamide, vanadyl-ribonucleoside complexes, macaloid, heparin,hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate,dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol,tris (2-carboxyethyl) phosphene hydrochloride, a divalent cation such asMg+2, Mn+2, Zn+2, Fe+2, Ca+2, Cu+2 and any combination thereof. c)ethylenediaminetetraacetic acid (EDTA); and d) one or more solvents;wherein the composition is free of separately added formaldehyde;wherein the amount of composition within the direct blood draw tubeprior to blood draw is less than about 1000 g/liter; and wherein thedirect blood draw tube including the composition and a blood samplelocated therein is storable at room temperature for at least about 14days without cell lysis and cell-free nucleic acid degradation of theblood sample. 32: The direct draw blood tube of claim 31, furtherincluding one or more formaldehyde releaser agents, wherein the one ormore formaldehyde releaser preservative agents are selected from thegroup consisting of: diazolidinyl urea, urea,dimethoylol-5,5dimethylhydantoin, dimethylol urea,2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxym ethylglycinate, 5-hydroxym ethoxym ethyl-1-1aza-3, 7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-1-1aza-3,7dioxabicyclo[3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3, 7dioxabicyclo[3.3.0]octane,quaternary adamantine and any combination thereof. 33: The direct blooddraw tube of claim 31, wherein the one or more enzyme inhibitors includebeta-mercaptoethanol and/or cysteine. 34: The direct draw blood tube ofclaim 31, wherein the direct blood draw tube is comprised of a polymericmaterial and/or an interior wall of the tube is coated or otherwisetreated with a polymeric coating to modify its surface characteristics.35: The direct draw blood tube of claim 34, wherein the polymericmaterial includes polyethylene terephthalate. 36: The direct blood drawtube of claim 35, wherein the composition is in a form of a gel or othersubstantially solid or highly viscous layer and wherein the compositionis re-dissolved or otherwise dispersed upon contact with the bloodsample. 37: The direct draw blood tube of claim 31, wherein the directdraw blood tube including the composition is storable at roomtemperature without deterioration of the composition. 38: The directblood draw tube of claim 35, wherein the one or more enzyme inhibitorsinclude beta-mercaptoethanol and/or cysteine. 39: A direct blood drawtube for stabilization of cell-free nucleic acid within a blood samplecomprising: a composition including: a) imidazolidinyl urea; b) one ormore of the following: i) one or more enzyme inhibitors; ii) one or moremetabolic inhibitors; and iii) one or more protease inhibitors c)ethylenediaminetetraacetic acid (EDTA); and d) one or more solvents;wherein the composition is free of separately added formaldehyde;wherein the amount of composition within the direct blood draw tubeprior to blood draw is less than about 1000 g/liter; and wherein thedirect blood draw tube including the composition and a blood samplelocated therein is storable at room temperature for at least about 14days without cell lysis and cell-free nucleic acid degradation of theblood sample.